Cellular levels and molecular dynamics simulations of estragole DNA adducts point at inefficient repair resulting from limited distortion of the double-stranded DNA helix

Abstract

Estragole, naturally occurring in a variety of herbs and spices, can form DNA adducts after bioactivation. Estragole DNA adduct formation and repair was studied in in vitro liver cell models, and a molecular dynamics simulation was used to investigate the conformation dependent (in)efficiency of N 2 -(trans-isoestragol-3′-yl)-2′-deoxyguanosine (E-3′-N 2 -dG) DNA adduct repair. HepG2, HepaRG cells, primary rat hepatocytes and CHO cells (including CHO wild-type and three NER-deficient mutants) were exposed to 50 μM estragole or 1′-hydroxyestragole and DNA adduct formation was quantified by LC-MS immediately following exposure and after a period of repair. Results obtained from CHO cell lines indicated that NER plays a role in repair of E-3′-N 2 -dG adducts, however, with limited efficiency since in the CHO wt cells 80% DNA adducts remained upon 24 h repair. Inefficiency of DNA repair was also found in HepaRG cells and primary rat hepatocytes. Changes in DNA structure resulting from E-3′-N 2 -dG adduct formation were investigated by molecular dynamics simulations. Results from molecular dynamics simulations revealed that conformational changes in double-stranded DNA by E-3′-N 2 -dG adduct formation are small, providing a possible explanation for the restrained repair, which may require larger distortions in the DNA structure. NER-mediated enzymatic repair of E-3′-N 2 -dG DNA adducts upon exposure to estragole will be limited, providing opportunities for accumulation of damage upon repeated daily exposure. The inability of this enzymatic repair is likely due to a limited distortion of the DNA double-stranded helix resulting in inefficient activation of nucleotide excision repair.

Molecular Dynamics and In Vitro Quantification of Safrole DNA Adducts Reveal DNA Adduct Persistence Due to Limited DNA Distortion Resulting in Inefficient Repair

“…In 1984, Phillips et al found that DNA adducts detected upon estragole, methyleugenol, and safrole exposure of B6C3F1 mice were still present 22 days after the final treatment. We recently corroborated the persistence of the major estragole DNA adduct (E-3′- N 2 -dG) with over 80% of the adduct level remaining in rat hepatocytes and human HepaRG cells after 72 h of repair …”

“…In line with this observation, we previously demonstrated that, when HepaRG cells and primary rat hepatocytes were exposed to the related alkenylbenzene estragole for 2 h, they appeared to contain still more than 70% of the formed DNA adducts upon a subsequent 72 h recovery period in the absence of the alkenylbenzene. This pointed at limited repair and resistance toward NER mediated DNA adduct elimination . Resistance toward NER mediated DNA adduct repair has also been reported for DNA adducts of some other genotoxic carcinogens including AFB1 FAPY adduct derived from aflatoxin, ALII- N 6 -dA adduct derived from aristolochic acids, and the polycyclic aromatic hydrocarbon (PAH) diol epoxide- N 6 -adenine DNA adduct formed upon exposure to fjord-type PAHs …”

“…The initial structure of S-3′- N 2 -dG was built using Molecular Operating Environment (MOE) software (version 2018.0101, Chemical Computing Group, Montreal), because no experimentally determined structure is available. The safrole adduct was added on the G6* of the B-type form DNA 11-mer base sequence used previously to study distortion of the dsDNA helix structure upon formation of the E-3′- N 2 -dG estragole adduct and the B­[a]­PDE- N 2 -dG adduct (Figure a). , The GROMOS force field 45A4 parameter set was used to describe molecular interactions . In a recent study, using AMBER force fields, including the bsc1 and OL15 modifications, simulations with longer simulation time could not provide a satisfactory description of the A/B equilibrium of the DNA, with a too strong emphasis on the B-type and hardly any occurrence of the A-type.…”

“…The transition between A-DNA and B-DNA is a basic element of DNA’s conformational repertoire, and it is important to investigate how the safrole adduct formation would affect the DNA structure in terms of the conformational equilibrium of the DNA. The GROMOS 45A4 force field allows for this flexibility and has previously successfully been used in a number of works in the literature. − In particular, we have also used this parameter set in our previous work on estragole adduct formation . The force field parameters for S-3′- N 2 -dG were derived based on analogy of similar functional groups in this force field and are supplied in Figure S1 in the Supporting Information.…”

“…Hydrogen bonding is defined by a minimum donor-hydrogen-acceptor angle of 135° and a maximum hydrogen-acceptor distance of 0.25 nm . DNA duplex helicoidal parameters were analyzed in the same way as described previously . The parameters evaluated in the analysis include six local base pair parameters (buckle, propeller, opening, shear, stretch, and stagger) and six base pair step parameters (rise, roll, shift, slide, and tilt).…”